Elemental analyses reveal distinct mineralization patterns in radular teeth of various molluscan taxa

The molluscan phylum is the second specious animal group with its taxa feeding on a variety of food sources. This is enabled by the radula, a chitinous membrane with embedded teeth, one important autapomorphy. Between species, radulae can vary in their morphology, mechanical, and chemical properties. With regard to chemical composition, some taxa (Polyplacophora and Patellogastropoda) were studied extensively in the past decades, due to their specificity to incorporate high proportions of iron, calcium, and silicon. There is, however, a huge lack of knowledge about radular composition in other taxa. The work presented aims at shedding light on the chemistry by performing energy-dispersive X-ray spectroscopy analyses on 24 molluscan species, thereof two Polyplacophora, two Cephalopoda, and 20 Gastropoda, which was never done before in such a comprehensiveness. The elements and their proportions were documented for 1448 individual, mature teeth and hypotheses about potential biomineralization types were proposed. The presented work additionally comprises a detailed record on past studies about the chemical composition of molluscan teeth, which is an important basis for further investigation of the radular chemistry. The found disparity in elements detected, in their distribution and proportions highlights the diversity of evolutionary solutions, as it depicts multiple biomineralization types present within Mollusca.

Elemental proportions. Summarizing all radulae studied (for mean, SD, and N see Supplementary   Table 1), we detect that Fe is present in the highest proportions, followed by Si, Ca, P, F, Na, Mg, S, Cl, Cu, and finally K. Of the 1448 areas studied, most of them contained Ca, followed by P, Mg, S, Cl, Na, Si, F, Fe, Cu, and finally K. Overall, the highest content of all analysed elements (sum, in atomic %, of the means of F, Na, Mg, Si, P, S, Cl, K, Ca, Fe, and Cu; see Supplementary Table 2 for values) was detected for the studied Patellogastropoda. This is followed by the Polyplacophora, Heterobranchia, Caenogastropoda, Cephalopoda, Vetigastropoda, and finally the Neritimorpha.

Composition-and biomineralization-types.
Overall, we detect that multiple composition-and biomineralization types are present within each individual species and also within individual teeth (see Fig. 2). Within Polyplacophora and Patellogastropoda we detected strong indications for the presence of the composition type I (containing Fe), II (Mg, Ca), III (Ca, P, Cl, F), IV (Si), OB (Na, S, K). In Cephalopoda, the types II, III, IV, V (Cu), and OB occur. In Vetigastropoda and Neritimorpha, the types II, III, IV, and OB were detected, in Vittina turrita-additionally the type I. In Caenogastropoda and Heterobranchia, the radular composition greatly varies between taxa. Overall, in Caenogastropoda, all composition types were found. However, in each species, the types II, III, and OB were always present, whereas Fe was only determined in Reymondia horei and Littorina littorea, Cu -in R. horei, and K -in Paramelania damoni, Cleopatra johnstoni, R. horei, Faunus ater, and L. littorea. C. johnstoni is the only species that seems to lack Ca. In Heterobranchia, the types II and OB were found in each species. The type III (apatite) is present in Onchidoris bilamellata, Aeolidia papillosa, Polycera quadrilineata, and Doris pseudoargus, but not in Cornu aspersum, as P, Cl, and F were not determined in this species. Si was only detected in C. aspersum and K in O. bilamellata.

Ingesta versus radular morphology and elemental proportions.
Morphology. The longest radulae were detected in species foraging on medium to solid ingesta, followed by solid-, medium-, soft-to-solid-, and finally soft-feeders with the shortest radulae (see Table 2 and Supplementary Figs. [28][29][30]. The largest radular area was calculated for medium-, medium-to-solid-, soft-to-solid-, solid-, and finally for the soft-feeders www.nature.com/scientificreports/ with the smallest area. Species foraging on medium-to-solid ingesta possess the highest quantity of tooth rows, followed by solid-, soft-to-solid-, medium-, and finally with the least quantity of tooth rows the soft-feeders.
All elements. In general, we detect that radulae of species foraging on solid ingesta possess the highest proportions of all studied elements, followed by species foraging on medium, medium-to-solid, soft-to-solid, and finally species feeding on soft ingesta.
Composition-type I. The highest Fe-proportions (means) were detected in the exclusively solid-, followed by the medium-to-solid-feeders. No Fe was detected for all other ingesta types.
Composition-type II and III. The highest proportions of Mg were detected in species foraging on medium ingesta, followed by solid-, soft-, medium-to-solid-, and finally soft-to-solid-feeders. Ca was detected in the highest proportions in the medium-, followed by the soft-, solid-, soft-to-solid-, and medium-to-solid-feeders. P was mainly found in species feeding on medium ingesta, followed by soft-to-solid-, solid-, medium-to-solid-, and finally soft-feeders. Cl was detected in the highest proportions in medium-feeders, followed by species feeding on soft-to-solid, soft, solid, and finally medium-to-solid ingesta. The highest proportions of F were found in radulae of medium-, followed by solid-, and finally medium-to-solid-feeders. In soft and soft-to-solid-feeders, no F was found. Table 2. Proportions of elements, radular length, area, quantity of tooth rows for the species foraging on certain ingesta types. N, quantity of teeth that contain the element, or quantity of radulae studied. www.nature.com/scientificreports/ Composition-type IV. The highest Si-content was detected in solid-, followed by medium-, medium-to-solid-, and finally soft-to-solid-feeders. No Si was found in species feeding on soft ingesta.
Composition-type V. The highest Cu proportions were detected in the solid-feeder Reymondia horei and less in the cephalopods foraging on soft-to-solid ingesta. All other radulae seem to lack Cu.
OB. S was detected in the highest proportions in species feeding on soft, followed by solid, soft-to-solid, medium-to-solid, and finally medium ingesta. The highest Na-proportions were detected in solid-, followed by the soft-, soft-to-solid-, medium-, and finally medium-to-solid-feeders. K was detected in the highest proportions in solid-, followed by medium-, medium-to-solid-, soft-to-solid-, and finally soft-feeders.
Correlations between parameters. In some cases we could detect correlations (please see Supplementary  Tables 4-16 for correlation coefficients). In general, radular area highly correlates with radular length and radular width. Additionally, in most cases, proportions of all elements highly correlate with each individual element. For individual elemental proportions, we here only highlight some, but the picture is rather puzzling. When all species are pooled together (see Supplementary Table 4) Ca and Cl, F and Ca, Fe and Ca, P and Ca, P and Cl, Si and Ca, Si and Fe correlate highly. For all soft ingesta feeders pooled together (see Supplementary Table 5) Mg and Na, K and P, Ca and P, Cl and P, Cl and K, Cl and Ca highly correlate. For all soft-to-solid ingesta feeders pooled together (see Supplementary Table 6) K and P, K and S, Ca and K, Cl and P, Cl and K highly correlate. For all medium ingesta feeders pooled together (see Supplementary Table 7) S and Si, K and Mg, Ca and P, F and P, F and Ca, Cl and S highly correlate. For all medium-to-solid ingesta feeders pooled together (see Supplementary  Table 8) Si and Na, Si and Mg, K and Na, Ca and P, Ca and K, F and P, F and K, F and Ca, Cl and P, Cl and Ca highly correlate. For all solid ingesta feeders pooled together (see Supplementary Table 9) Si and Mg, P and Si, S and Si, K and P, Ca and P, Cl and P correlate highly.
When the species studied are sorted by their taxonomic group we find, for most parameters no high correlations. However, for Caenogastropoda (see Supplementary Table 10) K and Si, F and P, F and K, F and Ca, Cl and P highly correlate. In Cephalopoda (see Supplementary Table 11) Cl and Ca correlate highly. In Heterobranchia (see Supplementary Table 12) K and Mg, Ca and P, F and P, Cl and Na are highly correlated. In Neritimorpha (see Supplementary Table 13) Si and Na, Si and Mg, Ca and P, Cl and P, Cl and Ca are highly correlated. In Patellogastropoda (see Supplementary Table 14) Si and Mg, P and Mg, S and P, K and Na, K and P, K and S, Ca and Mg, Ca and P highly correlate. In Polyplacophora (see Supplementary Table 15) P and Na, S and Mg, K and P, Fe and Si, F and K, Cl and Na are highly correlated. In Vetigastropoda (see Supplementary Table 16) S and Na highly correlate.
PCA on the individual parameters (elemental proportions, radular length, radular width, radular area, tooth rows) for all species pooled together detected no clustering (see Supplementary Fig. 31 A with highlighted systematic groups and B with highlighted ingesta categories).

Discussion
A detailed list of previous studies aimed at determining the chemical composition of the molluscan radula is provided in Supplementary Table 17. Most of the previous research has been done on the Polyplacophora with the focus exclusively on the dominant lateral teeth [for reviews see [33][34][35][36]80 ], except for one study on Lepidochitona cinerea determining the elemental composition of all tooth types 32 . Many of these analyses focused on the Fe biomineralization and the phase transformations during maturation [e.g. 23,29,[81][82][83][84][85][86][87][88][89] ]. Overall, in previous studies F, Na, Mg, Si, P, S, Cl, K, Ca, Fe, and Cu was detected in the dominant lateral teeth (= lateral teeth II) of Polyplacophora. For Lepidochitona cinerea, in our previous paper, we did not detect Cl, F, and Cu and in Acanthochitona fascicularis -no Si and Cu.
The following Fe proportions of mature dominant lateral teeth were previously determined in Polyplacophora: for A. fascicularis -59.2% 90 85,93 were previously detected in the dominant lateral teeth of chitons. S was also previously determined 83 . It is associated with the tanning of the organic matrix and with the appearance of proteins 82 . Additionally, 102 detected Zi, K, F, S, Na, and Cl in radular segments of Clavarizona 90 , Ca, P, Mg, S, Na, Zi, K, Al, Cu, and Si in radulae of Acanthopleura and Plaxiphora, and 83 Mg (with max ~ 5.5 weight %), K (with max ~ 1.0 weight %), Na (with max ~ 2.0 weight %), Si (with max ~ 1.0 weight %), Al (with max ~ 0.5 weight %), and S (with max ~ 0.8 weight %) in Cryptoplax. These elements, except for Zi and Cu, which were not found, occurred in smaller proportions (0-5%) in both Lepidochitona cinerea and Acanthochitona fascicularis. For the central, lateral I, and marginal teeth we detected less minerals than in the dominant lateral teeth in both species.
For the remaining gastropod taxa, only few analyses on the radular chemistry were conducted and usually the presence of elements, but not their proportions, could be determined. One of the earliest studies was done by 41 depicting results from Bergh, who performed complex chemical analyses of ashing and dissolving radulae from the Caenogastropods Charonia lampas (detecting P, Ca, and Fe), Lamellaria perspicua (detecting no Si), and Gibberulus gibberulus (probably detecting none of these elements, this is not clear) in different acids. Additionally, 41 presented his own results on the radulae of the Caenogastropod Tonna galea and the Heterobranch Helix nemoralis discovering P, Ca, and Fe in both by employing the same experiment. Sollas 42 was the first, who studied the radular chemistry in an elevated quantity of taxa, and 43 proceeded. Overall, their protocols are rather complex, involving analytical chemistry methods (ashing, staining, boiling, treating with acids, and using diffusion column) or physics (radula's refractive index). Sollas 42 42 determined Si and P. She detected Si in specimens collected during winter and phosphoric acid (P) in specimens collected during spring. 116 performed EDX analyses on five specimen of C. aspersum detecting Ca in all specimen and Si in one, even though specimens were also inventoried in spring depicting the inconsistency of elements embedded. For the Vetigastropoda (Haliotis tuberculata), 42 detected Si, Ca, and Fe.
The following species were studied by 42 and 43 , but for many species their results are contradictory. In the Caenogastropoda Littorina littorea 42 detected Mg, P, Ca, and Fe, whereas 43 found no Ca and no Fe. Nucella lapillus and Buccinum undatum were also studied by 42 , but the results are not clear from the publication, and 43 detected no Si and no Fe in both species. For the Vetigastropoda (Emarginula fissure and Calliostoma zizyphinum) 42 detected P, Ca, and Fe, whereas 43 determined the absence of Fe and Si. Then 117 and 26,118 were the first to close the existing gap in knowledge about the radular composition of Vetigastropoda. Gray 117 detected Na, Mg, Si, Cl, Ca, and Fe (EDX), and 26,118 Mg, Cl, Ca, and Fe (by EDX and inductively coupled plasma-optical emission spectrometry) in the limpet Megathura crenulata. Within Vetigastropoda, we detected Na, S, Cu, Si, P, Cl, Ca, and Mg; all of them in low proportions < 2%. Cu and S were not documented before, whereas Fe was detected in previous studies 117,118 . For the Neritimorpha, only one past study addresses the mineral content detecting S, Cl, K, Ca, Mg, Si, and Fe 119 . We additionally detected Na and P in Vittina turrita; all elements are abundant in very low proportions (< 4%). In the Caenogastropoda, we detected Fe, Mg, Ca, Cl, P, F, Si, Cu, S, Na, and K. Cu, F, Na, Si, S, and Cl were not determined before. In all species, proportions are < 6%. For the Heterobranchia, we detected more elements (Mg, Ca, Cl, P, F, Si, S, Na, K) than described in past publications [41][42][43]116 . Mg, Cl, F, S, Na, and K were not detected before. All elements are abundant at proportions < 15%.
Overall, the above data depicts that it is rather difficult to compare the percentages measured between studies, because in some weight percentages and in others atomic ratios were determined. Besides, methodology, sample preparation, and the analysed sample itself (whole radula or individual radular parts) differs. In addition, the presence and abundance of elements could potentially be influenced by the food available (e.g. plants containing or lacking Si) or by the chemistry of the saliva. In some taxa, specifically carnivorous gastropods, the saliva is acid [e.g. 120,121 ], so potentially the contact of the outermost radular teeth with the saliva leads to reduced proportions. Both ideas await further research.
The generally accepted hypothesis on radular mineralization evolution states that all gastropods -besides Patellogastropoda, Neritimorpha, and Vetigastropoda -probably lack Fe in the radula [e.g. [122][123][124] ]. However, Fe was detected previously in gastropod species [for Tonna galea, Charonia lampas, and Helix nemoralis see 41 , for Littorina littorea see 42 ] and our own analyses determined it in Reymondia horei and Littorina littorea. Thus, this means that iron is not lacking, rather its proportions are reduced in these gastropod lineages (see Fig. 1).
Previous studies relate the radular length to the ingesta type. Herbivorous taxa were found to possess longer radulae than carnivorous ones 125 . Littorinid species, feeding on algae covering rocks, were found to possess longer radulae than species feeding from plant surface [126][127][128][129][130] . For Patella species, it was determined that the radular length increases with increasing usage and wear 131 and, when algae are less abundant and the radula must thus be used more frequently to obtain the food necessary, its length increases 132 .
In general, we detected a similar pattern for the species studied here as the longest radulae with the highest quantity of tooth rows were found in species foraging on harder ingesta types (medium-to-solid, solid, medium) and the shortest ones in soft-substrate feeders. We, however, could not directly relate herbivory with longer radulae and carnivorous feeding with shorter ones. We additionally detected some relationship between radular length and proportions of elements (e.g. in Patella vulgata), so potentially more mineralized radulae are longer, because their maturation and mineralization requires more time and a longer contact to the overlain epithelia in the radular sac and mineralization zone. However, this does not seem to be the case for every species, as Lepidochitona cinerea and Acanthochitona fascicularis have relatively short heavily mineralized radulae. Thus, in these polyplacophoran species, the overlain epithelia can presumably incorporate more minerals at the same time or the radular replacement rate is faster in P. vulgata in contrast to the one in the Polyplacophora. Unfortunately, the radular replacement rate is known for few taxa: for Polyplacophorans (Acanthopleura, Plaxiphora, Patelloida, Mopalia), a rate of 0.36-0.80 rows per day was determined 90 www.nature.com/scientificreports/ described 135 . In Caenogastropoda, for Lacuna (Littorinidae), the rate of 3 rows/day 136 , for three Littorina species (Littorinidae) -5-6 rows/day depending on the temperature 2,135 , and for Pomatias elegans-5.02 rows/day 2 was determined. For Heterobranchia, the rate of 2.9 rows/day in Lymnaea stagnalis 137 , 5.02 rows/day in Agriolimax reticulatus 2 , 3.6 rows/day in adult Helix aspersa (= Cornu aspersum) was detected 2 . For Cepaea nemoralis, the whole radula was found to be renewed within 30-35 days 138 . Thus, in general, a higher degree of mineralization is inversely related to the higher replacement rate (teeth that possess larger proportions of minerals are replaced slower). However, radular replacement seems to depend on many factors, such as water temperature, metabolic rates, or age of animals 135,136,139,140 . Further studies on these questions are required.
In general, we detected that radulae of species, foraging on the solid ingesta, possess heavy mineralized teeth and species feeding on the soft ingesta show the smallest proportions. In biological materials, heterogeneities can have their origin in geometry, chemistry, and/or structure [for a review see 141 ]. In the dominant lateral teeth of chitons and limpets they have their origin in the distribution of the inorganic components and in the architecture of organic components 23,25,[27][28][29][30][31] . We have previously correlated the hardness and elasticity values in Lepidochitona cinerea with the iron and the calcium proportions 32 , which was previously also described for limpet teeth 24,104,108,142 and for other chitons 23,25,29,30 . For the paludomid gastropods, we previously measured elasticity modulus values ranging from 2 GPa at the tooth basis to 8 GPa in the cusp in solid substrate feeders, whereas soft substrate feeders possessed significantly softer teeth (4.6 GPa) 37,38,40 . In these species, we here detected inorganic elements in rather small proportions. We thus propose that specific cross-linking conditions of the chitin due to tanning 1 , fiber arrangement, and density 22,23,26,28,31,88,[143][144][145] rather cause the heterogeneities in mechanical properties. We previously also detected that the capability of wet teeth to rely on one another and to redistribute the mechanical stress increases the radula's resistance to structural failure in paludomid gastropods 146,147 . This altogether probably enables the feeding on harder ingesta types. Whether these mechanisms are also applicable for the other molluscan species, await further investigations.

Methods
Specimen studied and dissection. Mollusks were obtained from various sources (see Table 1  Species identification was reviewed by employing the relevant literature, the nomenclature and systematic position were checked using molluscabase.org. Not previously inventoried specimens were incorporated in the malacological collection of the ZMH, which is now part of the Leibniz-Institut für die Analyse des Biodiversitätswandels (LIB).
Overall, data from 72 adult specimens were analysed for this study. For each species, three adult specimens were selected, except for Histioteuthis spec. with two. We have chosen specimens of similar size per species, since the relationship between specimens' length and radular size is puzzling. Some previous studies relate both parameters 148 and others rather see a loose relationship or could not relate them for every species 125,149 . Additionally, the specimens chosen for each species were collected at the same time since seasonal dependencies in radular length were previously reported 18 . All data presented here is new, except for the elemental composition and radular morphology of Lepidochitona cinerea, which was taken from 32 . In this previous study, we analysed the ontogeny of the elemental composition and the mechanical parameters hardness and elasticity in three specimens of L. cinerea. For the present study, we included only the data from the working zone (the mature part) for the purpose of comparison between species.
Habitus images were either taken employing the Keyence Digital Microscope VHX-5000 (KEYENCE, Neu-Isenburg, Germany) or by using an iPad Pro (11 zoll; Apple Inc., Cupertino, USA) equipped with a 12-megapixel wide angle lens. Each specimen was dissected, the radula was carefully extracted by tweezers and then manually freed from surrounding tissue.

Scanning electron microscopy (SEM).
For images of the whole radula or the radular working zone, radulae (three per species, except for Histioteuthis spec. with two) were cleaned in an ultrasonic bath for 2-20 s and afterwards arranged on scanning electron microscopy (SEM) sample holders (see Supplementary Figs. 1-24). All radulae were first documented with the Keyence Digital Microscope VHX-5000 or VHX-7000 (KEYENCE, Neu-Isenburg, Germany). Here the length and width of each radula were measured and the quantity of tooth rows counted. From the length and width, the radular area was calculated. Two radulae per species were then visualized uncoated employing the Tabletop Microscope TM 4000 Plus (Hitachi, Tokyo, Japan) for more detailed images and one radula per species was coated and documented with the Zeiss LEO 1525 (One Zeiss Drive, Thornwood, USA) to receive images with a very high resolution (except for images of L. cinerea, they were taken from 150 , and of H. spec., as their radulae were documented uncoated and afterwards used for the EDX). Based www.nature.com/scientificreports/ on the morphology and arrangement of teeth, which were also categorized (e.g. central tooth, lateral tooth I, lateral tooth II, marginal tooth, inner teeth, outer teeth, etc.) according to their shape, size, and position on the membrane, radulae were assigned to different radular types (e.g. docogloss, isodont, rhipidogloss, etc.), if a suitable category could be determined from literature [e.g. 5,[151][152][153][154] ]. Then, the radulae, which were previously documented uncoated (two per species), were rewetted with 70% ethanol and loosened from the SEM sample holder and used for elemental analysis.

Elemental analysis (EDX).
Wet radulae were arranged on glass object slides (Carl Roth, Karlsruhe, Germany) with double-sided adhesive tape. They were positioned along their longitudinal axis so that the outermost teeth of one side were directly attached to the slide. The adjacent and more inner teeth were located above, followed by the central teeth, the inner teeth from the other side, and finally, on top, outer teeth again. Each radula was then dried for three days under ambient temperature and afterwards surrounded with a small, metallic ring ensuring an almost parallel sample surface. Epoxy resin (RECKLI EPOXI WST, RECKLI GmbH, Herne, Germany) was filled into the metallic ring and left polymerizing at room temperature for three days. This specific epoxy was chosen, since it does not infiltrate the teeth. Object slide and tape were then removed and, to receive longitudinal sections of each tooth, the embedded radulae were polished until the outer teeth were on display (controlled by examining the samples in the light microscope) using sandpapers of different roughness. Then samples were smoothed with aluminium oxide polishing powder suspension of 0.3 μm grainsize (PRESI GmbH, Hagen, Germany) on a polishing machine (Minitech 233/333, PRESI GmbH, Hagen, Germany). After polishing, the samples were cleaned from the polishing powder by an ultrasonic bath lasting five minutes. Samples were then coated with platinum (5 nm layer) and the elemental compositions of specific areas of the embedded teeth were examined employing the SEM Zeiss LEO 1525 (One Zeiss Drive, Thornwood, New York, USA) equipped with an Octane Silicon Drift Detector (SDD) (micro analyses system TEAM, EDAX Inc., New Jersey, USA) always using an acceleration voltage of 20 keV and the same settings (e.g. lens opening, working distance, etc.). Before measuring a sample the detector was always calibrated using cupper. We performed elemental mappings for test purposes, but for elements that are present in rather lower proportions, this method is not sensitive enough. We thus focused on the elemental analysis of small areas (10-400 μm 2 , depending on the tooth) trying to analyse the largest possible area.
The elements H (hydrogen), C (carbon), N (nitrogen), O (oxygen), Pt (platinum), Al (aluminium), Ca (calcium), Na (sodium), Mg (magnesium), Si (silicon), P (phosphorus), S (sulphur), Cl (chlorine), K (potassium), F (fluorine), Cu (copper), and Fe (iron) were detected and their proportions measured. We used the data of atomic ratio (atomic %) for this study. These values were received with two positions after the decimal point, lower proportions were not detectable with this method and therefore they were given as 0.00. We did not analyse and discuss the following elements, as they are either the elemental basis of chitin (H, C, N, O), the coating (Pt), or the polishing powder (Al, O).
After analysing the outer teeth, each sample was again polished and smoothened until the next tooth type was on display; cleaning procedure and EDX analyses were again performed. These steps were repeated until all teeth were analysed. In this study, we present the results of the radular working zone, which is not covered by epithelia. Thus, all teeth are mature. Overall, we use data of 1448 analysed teeth from 49 specimens.
Statistical analyses. All statistical analyses (mean, standard deviations) and visualizations with boxplots, pie charts, or trend lines were performed with JMP Pro, Version 14 (SAS Institute Inc., Cary, NC, 1989NC, -2007. Correlation coefficients and PCA were also conducted in JMP.

Composition-and biomineralization-types.
With EDX analysis, the proportions of the individual elements, present in a defined area, can be documented, whereas the specific bonding and structure of molecules cannot be analysed. However, from the percentile occurrence, in comparison with past studies on the radular chemical composition involving, we propose that the elements, detected here, are potentially part of the following molecules or minerals. These were assigned to different composition-or biomineralization-types: Category Type 1. Characterized by the presence of Fe. Potentially present in the form of magnetite as documented in polyplacophoran or goethite in limpets [e.g. 30,31,99,109,[155][156][157][158][159] ].
Category Type 2. Characterized by the presence of Mg and Ca. Elements are potentially involved in the protein packing, an increase in density of chitin fibres and in material stiffness as documented in limpet teeth 26 .
Category Type 4. Characterized by the presence of Si. Potentially present in the form of silica as documented in limpet teeth [e.g. 80,115 ].
Category Type 5. Characterized by the presence of Cu.
Category OB (organic bonds). The presence of Na, K, and S is often related to the protein bonding [e.g. 163